Not applicable.
Not applicable.
The present invention relates in part to isolated nucleic acid molecules (polynucleotides) which encode Ctenocephalides felis (flea) glutamate gated chloride channels. The present invention also relates to recombinant vectors and recombinant hosts which contain a DNA fragment encoding C. felis glutamate gated chloride channels, substantially purified forms of associated C. felis glutamate gated chloride channels, associated mutant proteins, and methods associated with identifying compounds which modulate associated Ctenocephalides felis glutamate gated chloride channels, which will be useful as insecticides.
Glutamate-gated chloride channels, or H-receptors, have been identified in arthropod nerve and muscle (Lingle et al, 1981, Brain Res. 212: 481-488; Horseman et al., 1988, Neurosci. Lett. 85: 65-70; Wafford and Sattelle, 1989, J. Exp. Bio. 144:449-462; Lea and Usherwood, 1973, Comp. Gen. Parmacol. 4: 333-350; and Cull-Candy, 1976, J. Physiol. 255:449-464).
Additionally, glutamate-gated chloride channels have been cloned from the soil nematode Caenorhabditis elegans (Cully et al., 1994, Nature 371: 707-711; see also U.S. Pat. No. 5,527,703) and Drosophila melanogaster (Cully et al., 1996, J. Biol. Chem. 271: 20187-20191).
Invertebrate glutamate-gated chloride channels are important targets for the widely used avermectin class of anthelmintic and insecticidal compounds. The avermectins are a family of macrocyclic lactones originally isolated from the actinomycete Streptomyces avermitilis. The semisynthetic avermectin derivative, ivermectin (22,23-dihydro-avermectin B1a), is used throughout the world to treat parasitic helminths and insect pests of man and animals. The avermectins remain the most potent broad spectrum endectocides exhibiting low toxicity to the host. After many years of use in the field, there remains little resistance to avermectin in the insect population. The combination of good therapeutic index and low resistance strongly suggests that the glutamate-gated chloride (GluCl) channels remain good targets for insecticide development.
It would be advantageous to identify additional invertebrate genes encoding encoding GluCl channels in order to allow screening to identify novel GluCl channel modulators that may have insecticidal, mitacidal and/or nematocidal activity for animal health or crop protection. The present invention addresses and meets these needs by disclosing isolated nucleic acid molecules which express a Ctenocephalides felis GluGl channel wherein expression of flea GluCl cRNA in Xenopus oocytes results in an active GluCl channel.
The present invention relates to isolated nucleic acid molecules (polynucleotides) which encode novel invertebrate GluCl channel proteins, especially nucleic acid molecules which encode a functional C. felis GluCl (herein, xe2x80x9cCfGluClxe2x80x9d) channel.
The present invention also relates to isolated nucleic acid fragments of CfGluCl which encode mRNA expressing a biologically active CfGluCl channel. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode cRNA which express a functional C. felis GluCl channel in a eukaryotic cell, such as Xenopus oocytes, so as to be useful for screening for agonists and/or antagonists of C. felis GluCl activity.
The isolated nucleic acid molecule of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA), including but not limited to messenger RNA (mRNA) encoding a biologically active C. felis GluCl channel and complementary RNA (cRNA) transcribed from a recombinant expression vector comprising a DNA molecule which encodes a full-length or biologically active portion of the full-length C. felis GluCl channel.
A preferred aspect of the present invention is disclosed in FIGS. 1A-B and SEQ ID NO:1, an isolated cDNA molecule encoding a C. felis GluCl channel, CfGluCl-1.
The present invention relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification, especially a nucleic acid molecule encoding a C. felis GluCl channel, CfGluCl, such as the cDNA molecule disclosed in FIGS. 1A-B and set forth in SEQ ID NO:1.
The present invention also relates to a substantially purified form of a C. felis GluCl channel protein and especially the C. felis GluCl channel disclosed in FIG. 2 and set forth in SEQ ID NO:2.
The present invention relates to a substantially purified membrane preparation which comprises a C. felis GluCl channel and is essentially free from contaminating proteins, including but not limited to other C. felis source proteins or host proteins from a recombinant cell which expresses CfGluCl. Especially preferred is a membrane preparation which comprises C. felis GluCl channel disclosed in FIG. 2 and set forth in SEQ ID NO:2. To this end, the present invention also relates to a substantially purified membrane preparation which is purified from a recombinant host, whether a recombinant eukaryotic or recombinant prokaryotic host, wherein a recombinant vector expresses a C. felis GluCl channel. Especially preferred is a membrane preparation which comprises a recombinant form of the C. felis GluCl channel, CfGluCl, disclosed in FIG. 2 and set forth in SEQ ID NO:2, referred to as CfGluCl-1.
The present invention also relates to biologically active fragments and/or mutants of a C. felis GluCl channel protein, including but not limited to the CfGluCl protein disclosed in FIG. 2 and set forth in SEQ ID NO:2, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for a biologically active channel which is useful in screening for agonists and/or antagonists of C. felis GluCl channel activity.
The present invention also relates to an isolated nucleic acid molecule (polynucleotide) which encodes a truncated form of the flea GluCl channel protein (herein, xe2x80x9ctr-CfGluClxe2x80x9d), as exemplified in FIG. 3 and set forth in SEQ ID NO:3. Co-expression of tr-CfGluCl in Xenopus oocytes with CfGluCl is shown to inhibit glutamate-gated channel activity.
The present invention also relates to isolated nucleic acid fragments of tr-CfGluCl-1 (SEQ ID NO:3) which encodes cRNA expressing a biologically active form of tr-CfGluCl, including but not limited to inhibition or promotion of CfGluCl channel activity in the target cell type. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations from the truncated form.
Again, any such truncated nucleic acid molecule (as compared to CfGluCl) may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA), including but not limited to messenger RNA (mRNA) or complementary RNA (cRNA) transcribed from a recombinant expression vector comprising a DNA molecule which encodes a truncated version of the full-length C. felis GluCl channel.
A preferred aspect of this portion of the invention is disclosed in FIGS. 3A-B and SEQ ID NO:4, an isolated cDNA molecule encoding a truncated version of the C. felis GluCl channel.
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification, especially a nucleic acid molecule encoding a truncated version of a C. felis GluCl channel such as the cDNA molecule disclosed in FIGS. 3A-B and set forth in SEQ ID NO:3.
The present invention also relates to a substantially purified form of a truncated version of the C. felis GluCl channel, trCfGluCl, and especially the truncated version of the C. felis GluCl channel, which is disclosed in FIG. 4 and as set forth in SEQ ID NO:4, referred to as trCfGluCl-1.
The present invention also relates to biologically active fragments and/or mutants of the truncated C. felis GluCl channel, trCfGluCl-1, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations.
It is an object of the present invention to provide an isolated nucleic acid molecule which encodes a novel form of a C. felis GluCl channel and biologically active fragments thereof which are derivatives of SEQ ID NO:2.
It is a further object of the present invention to provide the C. felis GluCl channel proteins or protein fragments encoded by the nucleic acid molecules referred to in the preceding paragraph.
It is a further object of the present invention to provide recombinant vectors and recombinant host cells which comprise a nucleic acid sequence encoding a C. felis GluCl channel or a biological equivalent thereof.
It is an object of the present invention to provide a substantially purified form of a C. felis GluCl channel or a biological equivalent thereof, as set forth in SEQ ID NO:2.
It is also an object of the present invention to provide a membrane preparation membrane preparation which comprises a C. felis GluCl channel and is essentially free from contaminating proteins. This membrane preparation includes, but is not limited to, a membrane preparation purified from a recombinant host.
It is an object of the present invention to provide for biologically active fragments and/or mutants of CfGluCl, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use.
It is an object of the present invention to provide a substantially purified form of CfGluCl-1, as set forth in SEQ ID NO:4.
It is an object of the present invention to provide for biologically active fragments and/or mutants of CfGluCl, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations.
As used herein, xe2x80x9cGluClxe2x80x9d refers to a glutamate-gated chloride channel.
As used herein, xe2x80x9cCfGluClxe2x80x9d refers to a biologically active form of a C. felis glutamate-gated chloride channel.
As used herein, xe2x80x9ccDNAxe2x80x9d refers to complementary DNA.
As used herein, xe2x80x9cmRNAxe2x80x9d refers to messenger RNA.
As used herein, xe2x80x9ccRNAxe2x80x9d refers to complementary RNA, transcribed from a recombinant cDNA template.